Bacterium producing L-glutamic acid and method for producing L-glutamic acid

ABSTRACT

L-Glutamic acid is produced by culturing a coryneform bacterium having L-glutamic acid producing ability, in which trehalose synthesis ability is decreased or deleted by, for example, disrupting a gene coding for trehalose-6-phosphate synthase, a gene coding for maltooligosyltrehalose synthase, or both of these genes to produce and accumulate L-glutamic acid in the medium, and collecting the L-glutamic acid from the medium.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a novel L-glutamic acidproducing bacterium and a method for producing L-glutamic acid byfermentation utilizing it. L-glutamic acid is an important amino acid asfoodstuffs, drugs and so forth.

[0003] 2. Description of the Related Art

[0004] Conventionally, L-glutamic acid is mainly produced byfermentative methods using so-called L-glutamic acid producingcoryneform bacteria belonging to the genus Brevibacterium,Corynebacterium or Microbacterium, or mutant strains thereof (Amino AcidFermentation, pp.195-215, Gakkai Shuppan Center, 1986).

[0005] It is known that, in the production of L-glutamic acid byfermentation, trehalose is also produced as a secondary product.Therefore, techniques have been developed for decomposing ormetabolizing the produced trehalose. Such techniques include the methodof producing an amino acid by fermentation using a coryneform bacteriumin which proliferation ability on trehalose is induced (Japanese PatentLaid-open (Kokai) No. 5-276935) and the method of producing amino acidby fermentation using a coryneform bacterium in which a gene coding fortrehalose catabolic enzyme is amplified (Korean Patent Publication (B1)No. 165836). However, it is not known how to suppress the formation oftrehalose itself in an L-glutamic acid producing bacterium.

[0006] In Escherichia coli, the synthesis of trehalose is catalyzed bytrehalose-6-phosphate synthase. This enzyme is known to be encoded byotsA gene. Further, it has been also reported that an otsAgene-disrupted strain of Escherichia coli can scarcely grow in ahyperosmotic medium (H. M. Glaever, et al., J. Bacteriol., 170(6),2841-2849 (1998)). However, the relationship between disruption of otsAgene and production of substances has not been known.

[0007] On the other hand, although the treY gene is known forBrevibacterium helvolum among bacteria belonging to the genusBrevibacterium bacteria, any otsA gene is not known for them. As forbacteria belonging to the genus Mycobacterium bacteria, there is known apathway via a reaction catalyzed by a product encoded by treS gene(trehalose synthase (TreS)), which gene is different from the otsA geneand treY gene, as a gene coding for a enzyme in trehalose biosynthesispathway (De Smet K. A., et al., Microbiology, 146 (1), 199-208 (2000)).However, this pathway utilizes maltose as a substrate and does notrelate to usual L-glutamic acid fermentation that utilizes glucose,fructose or sucrose as a starting material.

SUMMARY OF THE INVENTION

[0008] An object of the present invention is to improve productionefficiency of L-glutamic acid in L-glutamic acid production byfermentation using coryneform bacteria through suppression of theproduction of trehalose as a secondary product.

[0009] The inventors of the present invention assiduously studied inorder to achieve the aforementioned object. As a result, they found thatbacterium belonging to the genus Brevibacterium contained otsA gene andtreY gene like Mycobacterium tuberculosis, and the production efficiencyof L-glutamic acid was improved by disrupting at least one of thesegenes. Thus, they accomplished the present invention.

[0010] That is, the present invention provides the followings.

[0011] (1) A coryneform bacterium having L-glutamic acid producingability, wherein trehalose synthesis ability is decreased or deleted inthe bacterium.

[0012] (2) The coryneform bacteria according to (1), wherein thetrehalose synthesis ability is decreased or deleted by introducing amutation into a chromosomal gene coding for an enzyme in a trehalosesynthesis pathway or disrupting the gene.

[0013] (3) The coryneform bacteria according to (2), wherein the genecoding for the enzyme in trehalose synthesis pathway consists of a genecoding for trehalose-6-phosphate synthase, a gene coding formaltooligosyltrehalose synthase, or both of these genes.

[0014] (4) The coryneform bacteria according to (3), wherein the genecoding for trehalose-6-phosphate synthase codes for the amino acidsequence of SEQ ID NO: 30, and the gene coding formaltooligosyltrehalose synthase codes for the amino acid sequence of SEQID NO: 32.

[0015] (5) A method for producing L-glutamic acid comprising culturing acoryneform bacterium according to any one of (1) to (4) in a medium toproduce and accumulate L-glutamic acid in the medium, and collecting theL-glutamic acid from the medium.

[0016] (6) A DNA coding for a protein defined in the following (A) or(B):

[0017] (A) a protein having the amino acid sequence of SEQ ID NO: 30,

[0018] (B) a protein having an amino acid sequence of SEQ ID NO: 30including substitution, deletion, insertion or addition of one orseveral amino acid residues and having trehalose-6-phosphate synthaseactivity.

[0019] (7) A DNA according to (6), which is a DNA defined in thefollowing (a) or (b):

[0020] (a) a DNA containing a nucleotide sequence comprising at leastthe residues of nucleotide numbers 484-1938 in the nucleotide sequenceof SEQ ID NO: 29,

[0021] (b) a DNA hybridizable with a nucleotide sequence comprising atleast the residues of nucleotide numbers 484-1938 in the nucleotidesequence of SEQ ID NO: 29 under a stringent condition, showing homologyof 55% or more to the foregoing nucleotide sequence, and coding for aprotein having trehalose-6-phosphate synthase activity.

[0022] (8) A DNA coding for a protein defined in the following (A) or(B):

[0023] (A) a protein having the amino acid sequence of SEQ ID NO: 32,

[0024] (B) a protein having an amino acid sequence of SEQ ID NO: 32including substitution, deletion, insertion or addition of one orseveral amino acid residues and having maltooligosyltrehalose synthaseactivity.

[0025] (9) A DNA according to (8), which is a DNA defined in thefollowing (a) or (b):

[0026] (a) a DNA containing a nucleotide sequence comprising at leastthe residues of nucleotide numbers 82-2514 in the nucleotide sequence ofSEQ ID NO: 31,

[0027] (b) a DNA hybridizable with a nucleotide sequence comprising atleast the residues of nucleotide numbers 82-2514 in the nucleotidesequence of SEQ ID NO: 31 under a stringent condition, showing homologyof 60% or more to the foregoing nucleotide sequence, and coding for aprotein having maltooligosyltrehalose synthase activity.

[0028] The trehalose-6-phosphate synthase activity means an activity tocatalyze a reaction in which α,α-trehalose-6-phosphate and UDP areproduced from UDP-glucose and glucose-6-phosphate, and themaltooligosyltrehalose synthase activity means an activity to catalyze areaction in which maltotriosyltrehalose is produced from maltopentose.

[0029] According to the present invention, production efficiency ofL-glutamic acid in L-glutamic acid production by fermentation usingcoryneform bacteria can be improved through inhibition of the productionof trehalose as a secondary product.

PREFERREED EMBODIMENTS OF THE INVENTION

[0030] Hereafter, the present invention will be explained in detail.

[0031] The coryneform bacterium of the present invention is a coryneformbacterium having L-glutamic acid producing ability, in which trehalosesynthesis ability is decreased or deleted.

[0032] The coryneform bacteria referred to in the present inventioninclude the group of microorganisms defined in Bergey's Manual ofDeterminative Bacteriology, 8th edition, p.599 (1974), which are aerobicGram-positive rods having no acid resistance and no spore-formingability aerobic. They have hitherto been classified into the genusBrevibacterium, but united into the genus Corynebacterium at present(Int. J. Syst. Bacteriol., 41, 255 (1981)), and include bacteriabelonging to the genus Brevibacterium or Microbacterium closely relativeto the genus Corynebacterium. Examples of such coryneform bacteria arementioned below.

[0033]Corynebacterium acetoacidophilum

[0034]Corynebacterium acetoglutamicum

[0035]Corynebacterium alkanolyticum

[0036]Corynebacterium callunae

[0037]Corynebacterium glutamicum

[0038]Corynebacterium lilium (Corynebacterium glutamicum)

[0039]Corynebacterium melassecola

[0040]Corynebacterium thermoaminogenes

[0041]Corynebacterium herculis

[0042]Brevibacterium divaricatum (Corynebacterium glutamicum)

[0043]Brevibacterium flavum (Corynebacterium glutamicum)

[0044]Brevibacterium immariophilum

[0045]Brevibacterium lactofermentum (Corynebacterium glutamicum)

[0046]Brevibacterium roseum

[0047]Brevibacterium saccharolyticum

[0048]Brevibacterium thiogenitalis

[0049]Brevibacterium ammoniagenes (Corynebacterium ammoniagenes)

[0050]Brevibacterium album

[0051]Brevibacterium cerium

[0052]Microbacterium ammoniaphilum

[0053] Specifically, the following strains can be exemplified.

[0054]Corynebacterium acetoacidophilum ATCC 13870

[0055]Corynebacterium acetoglutamicum ATCC 15806

[0056]Corynebacterium alkanolyticum ATCC21511

[0057]Corynebacterium callunae ATCC 15991

[0058]Corynebacterium glutamicum ATCC 13020, 13032, 13060

[0059]Corynebacterium lilium (Corynebacterium glutamicum) ATCC 15990

[0060]Corynebacterium melassecola ATCC 17965

[0061]Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539)

[0062]Corynebacterium herculis ATCC13868

[0063]Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020

[0064]Brevibacterium flavum (Corynebacterium glutamicum) ATCC 13826,ATCC 14067

[0065]Brevibacterium immariophilum ATCC 14068

[0066]Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC13665, ATCC 13869

[0067]Brevibacterium roseum ATCC 13825

[0068]Brevibacterium saccharolyticum ATCC 14066

[0069]Brevibacterium thiogenitalis ATCC 19240

[0070]Brevibacterium ammoniagenes (Corynebacterium ammoniagenes) ATCC6871

[0071]Brevibacterium album ATCC 15111

[0072]Brevibacterium cerium ATCC 15112

[0073]Microbacterium ammoniaphilum ATCC 15354

[0074] The trehalose synthesis ability of such coryneform bacteria asmentioned above can be decreased or deleted by mutagenizing ordisrupting a gene coding for an enzyme in trehalose synthesis pathwayusing mutagenesis treatment or genetic recombination technique. Such amutation may be a mutation that suppresses transcription or translationof the gene coding for the enzyme in trehalose synthesis pathway, or amutation that causes elimination or decrease of an enzyme in trehalosesysthesis pathway. The enzyme in trehalose systhesis pathway may beexemplified by, for example, trehalose-6-phosphate synthase,maltooligosyltrehalose synthases, or both of these.

[0075] The disruption of a gene coding for an enzyme in trehalosesysthesis pathway can be performed by gene substitution utilizinghomologous recombination. A gene on a chromosome of a coryneformbacterium can be disrupted by transforming the coryneform bacterium withDNA containing a gene coding for an enzyme in trehalose systhesispathway modified so that a part thereof should be deleted and hence theenzyme in trehalose systhesis pathway should not normally function(deletion type gene), and allowing recombination between the deletiontype gene and a normal gene on the chromosome. Such gene disruption byhomologous recombination has already been established. To this end,there can be mentioned a method utilizing a linear DNA or a cyclic DNAthat does not replicate in coryneform bacteria and a method utilizing aplasmid containing a temperature sensitive replication origin. However,a method utilizing a cyclic DNA that does not replicate in coryneformbacteria or a plasmid containing a temperature sensitive replicationorigin is preferred.

[0076] The gene coding for an enzyme in trehalose systhesis pathway maybe exemplified by, for example, the otsA gene or treY gene, or it mayconsist of both of these. Since the nucleotide sequences of the otsAgene and treY gene of Brevibacterium lactofermentum and flanking regionsthereof have been elucidated by the present invention, those genes canbe easily obtained by preparing primers based on the sequences andperforming PCR (polymerase chain reaction, see White, T. J. et al.,Trends Genet., 5, 185 (1989)) using the primers and chromosomal DNA ofBrevibacterium lactofermentum as a template.

[0077] The nucleotide sequence comprising the otsA gene and thenucleotide sequence comprising the treY gene of Brevibacteriumlactofermentum obtained in the examples described later are shown in SEQID NOS: 29 and 31, respectively. Further, the amino acid sequencesencoded by these nucleotide sequences are shown in SEQ ID NOS: 30 and32, respectively.

[0078] The otsA gene and treY gene each may be one coding for a proteinincluding substitution, deletion, insertion or addition of one orseveral amino acids at one or a plurality of positions, provided thatthe activity of trehalose-6-phosphate synthase or maltooligosyltrehalosesynthase encoded thereby is not deteriorated. While the number of“several” amino acids differs depending on positions or types of aminoacid residues in the three-dimensional structure of the protein, it ispreferably 1-40, more preferably 1-20, further preferably 1-10.

[0079] A DNA coding for the substantially same protein astrehalose-6-phosphate synthase or maltooligosyltrehalose synthasedescribed above can be obtained by, for example, modifying each of thenucleotide sequences by, for example, the site-directed mutagenesismethod so that one or more amino acid residues at a specified siteshould involve substitution, deletion, insertion, addition or inversion.Such a DNA modified as described above may also be obtained by aconventionally known mutation treatment. The mutation treatment includesa method of treating DNA coding for trehalose-6-phosphate synthase ormaltooligosyltrehalose in vitro, for example, with hydroxylamine, and amethod for treating a microorganism, for example, a bacterium belongingto the genus Escherichia harboring a DNA coding fortrehalose-6-phosphate synthase or maltooligosyltrehalose withultraviolet irradiation or a mutating agent usually used for mutationtreatment such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrousacid.

[0080] The substitution, deletion, insertion, addition, or inversion ofnucleotide as described above also includes a naturally occurring mutantor variant on the basis of, for example, individual difference ordifference in species or genus of microorganisms that harbortrehalose-6-phosphate synthase or maltooligosyltrehalose.

[0081] A DNA coding for the substantially same protein astrehalose-6-phosphate synthase or maltooligosyltrehalose synthasedescribed above can be obtained by expressing such a DNA having amutation as described above in a suitable cell, and examining thetrehalose-6-phosphate synthase activity or maltooligosyltrehalosesynthase activity of the expression product.

[0082] A DNA coding for substantially the same protein astrehalose-6-phosphate synthase can also be obtained by isolating a DNAhybridizable with a DNA having, for example, a nucleotide sequencecorresponding to nucleotide numbers of 484-1938 of the nucleotidesequence shown in SEQ ID NO: 29 or a probe that can be prepared from thenucleotide sequence under a stringent condition, showing homology of 55%or more, preferably 65% or more, more preferably 75% or more, to theforegoing nucleotide sequence, and having trehalose-6-phosphate synthaseactivity from a DNA coding for trehalose-6-phosphate synthase having amutation or from a cell harboring it. Similarly, a DNA coding forsubstantially the same protein as maltooligosyltrehalose synthase canalso be obtained by isolating a DNA hybridizable with a DNA having, forexample, a nucleotide sequence corresponding to nucleotide numbers of82-2514 of the nucleotide sequence shown in SEQ ID NO: 31 or a probethat can be prepared from the nucleotide sequence under a stringentcondition, showing homology of 60% or more, preferably 70% or more, morepreferably 80% or more, to the foregoing nucleotide sequence, and havingmaltooligosyltrehalose synthase activity from a DNA coding formaltooligosyltrehalose synthase having a mutation or from a cellharboring it.

[0083] The “stringent condition” referred to herein is a condition underwhich so-called specific hybrid is formed, and non-specific hybrid isnot formed. It is difficult to clearly express this condition by usingany numerical value. However, for example, the stringent conditionincludes a condition under which DNA's having high homology, forexample, DNA's having homology of not less than 55%, preferably not lessthan 60%, are hybridized with each other, and DNA's having homologylower than the above level are not hybridized with each other.Alternatively, the stringent condition is exemplified by a conditionunder which DNA's are hybridized with each other at a salt concentrationcorresponding to an ordinary condition of washing in Southernhybridization, i.e., 1× SSC, 0.1% SDS, preferably 0.1× SSC, 0.1% SDS, at60° C.

[0084] As the probe, a partial sequence of each gene can also be used.Such a probe can be produced by PCR using oligonucleotides producedbased on the nucleotide sequence of each gene as primers and a DNAfragment containing each gene as a template. When a DNA fragment in alength of about 300 bp is used as the probe, the washing conditions forthe hybridization may consists of 50° C., 2× SSC and 0.1% SDS.

[0085] Genes hybridizable under such conditions as described aboveinclude those having a stop codon generated in a coding region of thegenes, and those having no activity due to mutation of active center.However, such mutants can be easily removed by ligating each of thegenes with a commercially available expression vector, and measuringtrehalose-6-phosphate synthase activity or maltooligosyltrehalosesynthase activity.

[0086] When an otsA gene or treY gene is used for the disruption ofthese genes on chromosomes of coryneform bacteria, the encodedtrehalose-6-phosphate synthase or maltooligosyltrehalose synthase arenot required to have their activities. Further, the otsA gene or treYgene used for the gene disruption may be a gene derived from anothermicroorganism, so long as they can undergo homologous recombination withthese genes of coryneform bacteria. For example, an otsA gene ofbacterium belonging to the genus Escherichia or Mycobacterium, treY geneof bacterium belonging to the genus Arthrobacter, Brevibacteriumhelvolum, or bacterium belonging to the genus Rhizobium can bementioned.

[0087] A deletion type gene of the otsA gene or treY gene can beprepared by excising a certain region with restriction enzyme(s) from aDNA fragment containing one of these genes or a part of them to deleteat least a part of coding region or an expression regulatory sequencesuch as promoter.

[0088] Further, a deletion type gene can also be obtained by performingPCR using primers designed so that a part of gene should be deleted.Furthermore, a deletion type gene may be one obtained by singlenucleotide mutation, for example, a frame shift mutation.

[0089] Gene disruption of the otsA gene will be explained hereafter.Gene disruption of the treY gene can be performed similarly.

[0090] An otsA gene on a host chromosome can be replaced with a deletiontype otsA gene as follows. That is, a deletion type otsA gene and amarker gene for resistance to a drug, such as kanamycin,chloramphenicol, tetracycline and streptomycin, are inserted into aplasmid that cannot autonomously replicate in coryneform bacteria toprepare a recombinant DNA. A coryneform bacterium can be transformedwith the recombinant DNA, and the transformant strain can be cultured ina medium containing the drug to obtain a transformant strain in whichthe recombinant DNA was introduced into chromosomal DNA. Alternatively,such a transformant strain can be obtained by using a temperaturesensitive plasmid as the plasmid, and culturing the transformants at atemperature at which the temperature sensitive plasmid cannot replicate.

[0091] In a strain in which the recombinant DNA is incorporated into achromosome as described above, the recombinant DNA causes recombinationwith an otsA gene sequence that originally exists on the chromosome, andtwo of fused genes comprising the chromosomal otsA gene and the deletiontype otsA gene are inserted into the chromosome so that other portionsof the recombinant DNA (vector portion and drug resistance marker gene)should be interposed between them.

[0092] Then, in order to leave only the deletion type otsA gene on thechromosomal DNA, one copy of the otsA gene is eliminated from thechromosomal DNA together with the vector portion (including the drugresistance marker gene) by recombination of two of the otsA genes. Inthat case, the normal otsA gene is left on the chromosomal DNA and thedeletion type otsA gene is excised, or conversely, the deletion typeotsA gene is left on the chromosomal DNA and the normal otsA gene isexcised. It can be confirmed which type of the gene is left on thechromosomal DNA by investigating structure of the otsA gene on thechromosome by PCR, hybridization or the like.

[0093] The coryneform bacterium used for the present invention may haveenhanced activity of an enzyme that catalyzes the biosynthesis ofL-glutamic acid in addition to the deletion or decrease of trehalosesynthesis ability. Examples of the enzyme that catalyzes thebiosynthesis of L-glutamic acid include glutamate dehydrogenase,glutamine synthetase, glutamate synthase, isocitrate dehydrogenase,aconitate hydratase, citrate synthase, pyruvate carboxylase,phosphoenolpyruvate carboxylase, phosphoenolpyruvate synthase, enolase,phosphoglyceromutase, phosphoglycerate kinase,glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase,fructose bisphosphate aldolase, phosphofructokinase, glucose phosphateisomerase and so forth.

[0094] Further, in the coryneform bacterium used for the presentinvention, an enzyme that catalyzes a reaction for generating a compoundother than L-glutamic acid by branching off from the biosyntheticpathway of L-glutamic acid may be declined or made deficient. Examplesof such an enzyme include α-ketoglutarate dehydrogenase, isocitratelyase, phosphate acetyltransferase, acetate kinase, acetohydroximatesynthase, acetolactate synthase, formate acetyltransferase, lactatedehydrogenase, L-glutamate decarboxylase, 1-pyrroline dehydrogenase andso forth.

[0095] Furthermore, by introducing a temperature sensitive mutation fora biotin activity inhibiting substance such as surface active agentsinto a coryneform bacterium having L-glutamic acid producing ability,the bacterium becomes to be able to produce L-glutamic acid in a mediumcontaining an excessive amount of biotin in the absence of a biotinactivity inhibiting substance (see WO96/06180). As such a coryneformbacterium, the Brevibacterium lactofermentum AJ13029 strain disclosed inWO96/06180 can be mentioned. The AJ13029 strain was deposited at theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology (currently, the independentadministrative corporation, National Institute of Advanced IndustrialScience and Technology, International Patent Organism Depositary (ChuoDai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postalcode: 305-5466) on Sep. 2, 1994, and received an accession number ofFERM P-14501. Then, it was transferred to an international deposit underthe provisions of the Budapest Treaty on Aug. 1, 1995, and received anaccession number of FERM BP-5189.

[0096] When a coryneform bacterium having L-glutamic acid producingability, in which trehalose synthesis ability is decreased or deleted,is cultured in a suitable medium, L-glutamic acid is accumulated in themedium.

[0097] The medium used for producing L-glutamic acid is a usual mediumthat contains a carbon source, a nitrogen source, inorganic ions andother organic trace nutrients as required. As the carbon source, it ispossible to use sugars such as glucose, lactose, galactose, fructose,sucrose, maltose, blackstrap molasses and starch hydrolysate; alcoholssuch as ethanol and inositol; or organic acids such as acetic acid,fumaric acid, citric acid and succinic acid.

[0098] As the nitrogen source, there can be used inorganic ammoniumsalts such as ammonium sulfate, ammonium nitrate, ammonium chloride,ammonium phosphate and ammonium acetate, ammonia, organic nitrogen suchas peptone, meat extract, yeast extract, corn steep liquor and soybeanhydrolysate, ammonia gas, aqueous ammonia and so forth.

[0099] As the inorganic ions (or sources thereof), added is a smallamount of potassium phosphate, magnesium sulfate, iron ions, manganeseions and so forth. As for the organic trace nutrients, it is desirableto add required substances such as vitamin B₁, yeast extract and soforth in a suitable amount as required.

[0100] The culture is preferably performed under an aerobic conditionperformed by shaking, stirring for aeration or the like for 16 to 72hours. The culture temperature is controlled to be at 30° C. to 45° C.,and pH is controlled to be 5 to 9 during the culture. For suchadjustment of pH, inorganic or organic acidic or alkaline substances,ammonia gas and so forth can be used.

[0101] Collection of L-glutamic acid from fermentation broth can beperformed by, for example, methods utilizing ion exchange resins,crystallization and so forth. Specifically, L-glutamic acid can beadsorbed on an anion exchange resin and isolated from it, orcrystallized by neutralization.

EXAMPLES

[0102] Hereafter, the present invention will be explained morespecifically with reference to the following examples.

Example 1 Construction of otsA Gene-disrupted Strain of Brevibacteriumlactofermentum

[0103] <1> Cloning of otsA Gene

[0104] Since otsA gene of Brevibacterium lactofermentum was not known,it was obtained by utilizing a nucleotide sequence of otsA gene ofanother microorganism for reference. The otsA genes of Escherichia andMycobacterium had been hitherto elucidated for their entire nucleotidesequences (Kaasen I., et al., Gene, 145 (1), 9-15 (1994); De Smet K. A.,et al., Microbiology, 146 (1), 199-208 (2000)). Therefore, referring toan amino acid sequence deduced from these nucleotide sequences, DNAprimers P1 (SEQ ID NO: 1) and P2 (SEQ ID NO: 2) for PCR were synthesizedfirst. The DNA primers P1 and P2 corresponded to the regions of thenucleotide numbers of 1894-1913 and 2531-2549 of the nucleotide sequenceof the otsA gene of Escherichia coli (GenBank accession X69160),respectively. They also corresponded to the regions of the nucleotidenumbers 40499-40518 and 41166-41184 of the otsA gene of Mycobacteriumtuberculosis (GenBank accession Z95390), respectively.

[0105] Then, PCR was performed by using the primers P1 and P2 andchromosomal DNA of Brevibacterium lactofermentum ATCC 13869 as atemplate with a cycle consisting of reactions at 94° C. for 0.5 minute,50° C. for 0.5 minute and 72° C. for 4 minutes, which was repeated for30 cycles. As a result, a substantially single kind of amplifiedfragment of about 0.6 kbp was obtained. This amplified fragment wascloned into a plasmid vector pCR2.1 by using “Original TA Cloning Kit”produced by Invitrogen to obtain pCotsA. Then, the nucleotide sequenceof the cloned fragment was determined.

[0106] Based on the nucleotide sequence of the partial fragment of otsAgene obtained as described above, DNA primers P10 (SEQ ID NO: 8) and P12(SEQ ID NO: 10) were newly synthesized, and unknown regions flanking tothe partial fragment was amplified by “inverse PCR” (Triglia, T. et al.,Nucleic Acids Res., 16, 81-86 (1988); Ochman H., et al., Genetics, 120,621-623 (1988)). The chromosomal DNA of Brevibacterium lactofermentumATCC 13869 was digested with a restriction enzyme BamHI, BglII, ClaI,HindIII, KpnI, MluI, MunL, SalI or XhoI, and self-ligated by using T4DNA ligase (Takara Shuzo). By using resultant DNA as a template and theDNA primers P10 and P12, PCR was performed with a cycle consisting ofreactions at 94° C. for 0.5 minute, 55° C. for 1 minute and 72° C. for 4minutes, which was repeated for 30 cycles. As a result, when ClaI orBglII was used as the restriction enzyme, an amplified fragment of 4 kbpwas obtained for each case. The nucleotide sequences of these amplifiedfragments were directly determined by using the DNA primers P5 to P9(SEQ ID NOS: 3-7) and P11 to P15 (SEQ ID NOS: 9-13). Thus, the entirenucleotide sequence of otsA gene of Brevibacterium lactofermentum ATCC13869 was determined as shown in SEQ ID NO: 29. The amino acid sequenceencoded by this nucleotide sequence is shown in SEQ ID NOS: 29 and 30.

[0107] When homology of the sequence of the aforementioned otsA gene wasdetermined with respect to the otsA gene of Escherichia coli (GenBankaccession X69160) and the otsA gene of Mycobacterium tuberculosis(GenBank accession Z95390), the nucleotide sequence showed homologies of46.3% and 55.9%, respectively, and the amino acid sequence showedhomologies of 30.9% and 51.7%, respectively. The homologies werecalculated by using software, “GENETIX-WIN” (Software Development),based on the Lipman-Person method (Science, 227, 1435-1441 (1985)).

[0108] <2> Preparation of Plasmid for otsA Gene Disruption

[0109] In order to examine presence or absence of improvement effect inL-glutamic acid productivity by disruption of a gene coding for anenzyme in trehalose biosysthesis pathway in coryneform bacteria, aplasmid for otsA gene disruption was produced. A plasmid for otsA genedisruption was produced as follows. PCR was performed by using theplasmid pCotsA previously constructed in the cloning of the otsA gene asa template and the primers P29 (SEQ ID NO: 33) and P30 (SEQ ID NO: 34)comprising ClaI site with a cycle consisting of reactions at 94° C. for0.5 minute, 55° C. for 0.5 minute and 72° C. for 8 minutes, which wasrepeated for 30 cycles. The amplified fragment was digested with ClaI,blunt-ended by using T4 DNA polymerase (Takara Shuzo), and self-ligatedby using T4 ligase (Takara Shuzo) to construct a plasmid pCotsACcontaining the otsA gene having a frame shift mutation (1258-1300thnucleotides of SEQ ID NO: 29 were deleted) at an approximately centralpart thereof.

[0110] <3> Preparation of otsA Gene-disrupted Strain

[0111] By using the plasmid pCotsAC for gene disruption, a L-glutamicacid producing bacterium, Brevibacterium lactofermentum ATCC 13869, wastransformed by the electric pulse method, and transformants wereselected as to the ability to grow in CM2B medium containing 20 mg/L ofkanamycin. Because the plasmid pCotsAC for otsA gene disruption did nothave a replication origin that could function in Brevibacteriumlactofermentum, resultant transformants obtained by using the plasmidsuffered homologous recombination occurred between the otsA genes on thechromosome of Brevibacterium lactofermentum and the plasmid pCotsAC forgene disruption. From the homologous recombinant strains obtained asdescribed above, strains in which the vector portion of the plasmidpCotsAC for gene disruption was eliminated due to re-occurrence ofhomologous recombination were selected based on acquired kanamycinsensitivity as a marker.

[0112] From the strains obtained as described above, a strain introducedwith the desired frame shift mutation was selected. Selection of such astrain was performed by PCR using chromosomal DNA extracted from astrain that became kanamycin sensitive as a template and the DNA primersP8 (SEQ ID NO: 14) and P13 (SEQ ID NO: 11) with a cycle consisting ofreactions at 94° C. for 0.5 minute, 55° C. for 0.5 minute and 72° C. for1 minutes, which was repeated for 30 cycles, and sequencing of theobtained amplified fragment using the DNA primer P8 to confirmdisfunction of the otsA gene due to introduction of frame shiftmutation. The strain obtained as described above was designated as AOAstrain.

Example 2 Construction of treY Gene-disrupted Strain

[0113] <1> Cloning of treY Gene

[0114] Since treY gene of Brevibacterium lactofermentum was not known,it was obtained by using nucleotide sequences of treY genes of the othermicroorganisms for reference. The nucleotide sequences of treY geneswere hitherto elucidated for the genera Arthrobacter, Brevibacterium andRhizobium (Maruta K., et al., Biochim. Biophys. Acta, 1289 (1), 10-13(1996); Genbank accession AF039919; Maruta K., et al., Biosci.Biotechnol. Biochem., 60 (4), 717-720 (1996)). Therefore, referring toan amino acid sequence deduced from these nucleotide sequences, the PCRDNA primers P3 (SEQ ID NO: 14) and P4 (SEQ ID NO: 15) were synthesizedfirst. The DNA primers P3 and P4 correspond to the regions of thenucleotide numbers of 975-992 and 2565-2584 of the nucleotide sequenceof the treY gene of Arthrobacter species (GenBank accession D63343),respectively. Further, they correspond to the regions of the nucleotidenumbers 893-910 and 2486-2505 of the treY gene of Brevibacteriumhelvolum (GenBank accession AF039919), respectively. Furthermore, theycorrespond to the regions of the nucleotide numbers of 862-879 and2452-2471 of treY gene of Rhizobium species (GenBank accession D78001).

[0115] Then, PCR was performed by using the primers P3 and P4 andchromosomal DNA of Brevibacterium lactofermentum ATCC13869 as a templatewith a cycle consisting of reactions at 94° C. for 0.5 minute, 55° C.for 0.5 minute and 72° C. for 2 minutes, which was repeated for 30cycles. As a result, a substantially single kind of an amplifiedfragment of about 1.6 kbp was obtained. This amplified fragment wascloned into a plasmid vector pCR2.1 by using “Original TA Cloning Kit”produced by Invitrogen. Then, the nucleotide sequence was determined forabout 0.6 kb.

[0116] Based on the nucleotide sequence of the partial fragment of treYgene obtained as described above, the DNA primers P16 (SEQ ID NO: 16)and P26 (SEQ ID NO: 26) were newly synthesized, and unknown regionsflanking to the partial fragment was amplified by “inverse PCR”(Triglia, T. et al., Nucleic Acids Res., 16, 81-86 (1988); Ochman H., etal., Genetics, 120, 621-623 (1988)). The chromosomal DNA ofBrevibacterium lactofermentum ATCC 13869 was digested with a restrictionenzyme BamHI, HindIII, SalI or XhoI, and self-ligated by using T4 DNAligase (Takara Shuzo). By using this as a template and the DNA primersP16 and P26, PCR was performed with a cycle consisting of reactions at94° C. for 0.5 minute, 55° C. for 1 minute and 72° C. for 4 minutes,which was repeated for 30 cycles. As a result, when HindIII or SalI wasused as the restriction enzyme, an amplified fragment of 0.6 kbp or 1.5kbp was obtained, respectively. The nucleotide sequences of theseamplified fragments were directly determined by using the DNA primersP16 to P28 (SEQ ID NOS: 16-28). Thus, the entire nucleotide sequence oftreY gene of Brevibacterium lactofermentum ATCC 13869 was determined asshown in SEQ ID NO: 31. The amino acid sequence encoded by thisnucleotide sequence is shown in SEQ ID NOS: 31 and 32.

[0117] When homology of the sequence of the aforementioned treY gene wasdetermined with respect to the treY gene of Arthrobacter sp. (GenBankaccession D63343), treY gene of Brevibacterium helvolum (GenBankaccession AF039919) and treY gene of Rhizobium sp. (GenBank accessionD78001), the nucleotide sequence showed homologies of 52.0%, 52.3% and51.9%, respectively, and the amino acid sequence showed homologies of40.9%, 38.5% and 39.8%, respectively. The homologies were calculated byusing software, “GENETIX-WIN” (Software Development), based on theLipman-Person method (Science, 227, 1435-1441 (1985)).

[0118] <2> Preparation of Plasmid for Trey Gene Disruption

[0119] In order to examine presence or absence of improvement effect inL-glutamic acid productivity by disruption of the gene coding for theenzyme in trehalose biosysthesis pathway in coryneform bacteria, aplasmid for treY gene disruption was produced. First, PCR was performedby using the primers P17 (SEQ ID NO: 17) and P25 (SEQ ID NO: 25) and thechromosomal DNA of ATCC 13869 as a template with a cycle consisting ofreactions at 94° C. for 0.5 minute, 60° C. for 0.5 minute and 72° C. for2 minutes, which was repeated for 30 cycles. The amplified fragment wasdigested with EcoRI and ligated to pHSG299 (Takara Shuzo) digested withEcoRI by using T4 DNA ligase (Takara Shuzo) to obtain a plasmid pHtreY.Further, this pHtreY was digested with AflII (Takara Shuzo), blunt-endedby using T4 DNA polymerase (Takara Shuzo), and self-ligated by using T4ligase (Takara Shuzo) to construct a plasmid pHtreYA containing the treYgene having a frame shift mutation (four nucleotides were inserted afterthe 1145th nucleotide in the sequence of SEQ ID NO: 31) at anapproximately central part thereof.

[0120] <3> Preparation of treY Gene-disrupted Strain

[0121] By using the plasmid pCtreYA for gene disruption, a L-glutamicacid producing bacterium, Brevibacterium lactofermentum ATCC 13869, wastransformed by the electric pulse method, and transformants wereselected as to the ability to grow in CM2B medium containing 20 mg/L ofkanamycin. Because the plasmid pCtreYA for treY gene disruption does nothave a replication origin that could function in Brevibacteriumlactofermentum, the transformants obtained by using the plasmid sufferedrecombination occurred between the treY genes on the Brevibacteriumlactofermentum chromosome and the plasmid pCtreYA for gene disruption.From the homologous recombinant strains obtained as described above,strains in which the vector portion of the plasmid pCtreYA for genedisruption was eliminated due to re-occurrence of homologousrecombination were selected based on acquired kanamycin sensitivity as amarker.

[0122] From the strains obtained as described above, a strain introducedwith the desired frame shift mutation was selected. Selection of such astrain was performed by PCR using the DNA primers P19 (SEQ ID NO: 19)and P25 (SEQ ID NO: 25) with a cycle consisting of reactions at 94° C.for 0.5 minute, 55° C. for 0.5 minute and 72° C. for 1.5 minutes, whichwas repeated for 30 cycles, and sequencing the obtained fragment usingthe DNA primer P21 or P23 to confirm dysfunction of the trey gene due tointroduction of frame shift mutation. The strain obtained as describedabove was designated as ΔTA strain.

Example 3 Evaluation of L-glutamic Acid Producing Ability of ΔOA Strainand ΔTA Strain

[0123] The ATCC 13869 strain, AOA strain and ATA strain were eachcultured for producing L-glutamic acid as follows. Each of these strainswas refreshed by culturing it on a CM2B plate medium, and each refreshedstrain was cultured in a medium containing 80 g of glucose, 1 g ofKH₂PO₄, 0.4 g of MgSO₄, 30 g of (NH₄)₂SO₄, 0.01 g of FeSO₄.7H₂O, 0.01 gMnSO₄.7H₂O, 15 ml of soybean hydrolysate solution, 200 μg of thiaminhydrochloride, 3 μg of biotin and 50 g of CaCO₃ in 1 L of pure water(adjusted to pH 8.0 with KOH) at 31.5° C. After the culture, amount ofL-glutamic acid accumulated in the medium and absorbance at 620 nm ofthe culture broth diluted 51 times were measured. The results are shownin Table 1.

[0124] The Brevibacterium lactofermentum strains of which otsA gene ortreY gene was disrupted showed growth in a degree similar to that of theparent strain, and in addition, increased L-glutamic acid productioncompared with the parent strain.

Table 1

[0125] TABLE 1 Strain OD₆₂₀ (x51) L-Glutamic acid (g/L) Yield (%) ATCC13869 0.930 40.2 48.4 ΔOA 1.063 43.8 52.8 ΔTA 0.850 45.6 54.9

[0126] (Explanation of Sequence Listing)

[0127] SEQ ID NO: 1: Primer P1 for amplification of otsA

[0128] SEQ ID NO: 2: Primer P2 for amplification of otsA

[0129] SEQ ID NO: 3: Primer P5

[0130] SEQ ID NO: 4: Primer P6

[0131] SEQ ID NO: 5: Primer P7

[0132] SEQ ID NO: 6: Primer P8

[0133] SEQ ID NO: 7: Primer P9

[0134] SEQ ID NO: 8: Primer P10

[0135] SEQ ID NO: 9: Primer P11

[0136] SEQ ID NO: 10: Primer P12

[0137] SEQ ID NO: 11: Primer P13

[0138] SEQ ID NO: 12: Primer P14

[0139] SEQ ID NO: 13: Primer P15

[0140] SEQ ID NO: 14: Primer P3 for amplification of treY

[0141] SEQ ID NO: 15: Primer P4 for amplification of treY

[0142] SEQ ID NO: 16: Primer P16

[0143] SEQ ID NO: 17: Primer P17

[0144] SEQ ID NO: 18: Primer P18

[0145] SEQ ID NO: 19: Primer P19

[0146] SEQ ID NO: 20: Primer P20

[0147] SEQ ID NO: 21: Primer P21

[0148] SEQ ID NO: 22: Primer P22

[0149] SEQ ID NO: 23: Primer P23

[0150] SEQ ID NO: 24: Primer P24

[0151] SEQ ID NO: 25: Primer P25

[0152] SEQ ID NO: 26: Primer P26

[0153] SEQ ID NO: 27: Primer P27

[0154] SEQ ID NO: 28: Primer P28

[0155] SEQ ID NO: 29: Nucleotide sequence of otsA gene

[0156] SEQ ID NO: 30: Amino acid sequence of OtsA

[0157] SEQ ID NO: 31: Nucleotide sequence of treY gene

[0158] SEQ ID NO: 32: Amino acid sequence of TreY

[0159] SEQ ID NO: 33: Primer P29

[0160] SEQ ID NO: 34: Primer P30

1 34 1 20 DNA Artificial Sequence Synthetic DNA 1 canathggnt tyttyytnca20 2 19 DNA Artificial Sequence Synthetic DNA 2 canarrttca tnccrtcnc 193 23 DNA Artificial Sequence Synthetic DNA 3 gaatcatcca tataagatcc ggc23 4 24 DNA Artificial Sequence Synthetic DNA 4 tagctttgta gttgttgctaaccg 24 5 24 DNA Artificial Sequence Synthetic DNA 5 agcgaacttgaggtttactt cccg 24 6 24 DNA Artificial Sequence Synthetic DNA 6tgctggttcc tggcattttg cgcc 24 7 20 DNA Artificial Sequence Synthetic DNA7 tcgaacaatc tcttcacgcc 20 8 21 DNA Artificial Sequence Synthetic DNA 8gaatcccacc aaatctgcgc c 21 9 20 DNA Artificial Sequence Synthetic DNA 9tgatgttgaa atgtttgggg 20 10 20 DNA Artificial Sequence Synthetic DNA 10gatgtcatgc tggttacgcc 20 11 22 DNA Artificial Sequence Synthetic DNA 11caaagcacca gtgccgtcgc gg 22 12 24 DNA Artificial Sequence Synthetic DNA12 tgttcgtttt cattcgcgtt gccg 24 13 24 DNA Artificial Sequence SyntheticDNA 13 atagtttcct ggattgtttg gcgc 24 14 18 DNA Artificial SequenceSynthetic DNA 14 caraayccnt ggtggtgg 18 15 20 DNA Artificial SequenceSynthetic DNA 15 ggncgncgrt trtcnggrtc 20 16 20 DNA Artificial SequenceSynthetic DNA 16 cgagctcttc attgatggcg 20 17 20 DNA Artificial SequenceSynthetic DNA 17 gcagctacac acgagttggg 20 18 20 DNA Artificial SequenceSynthetic DNA 18 gcaacaccta aatggttggg 20 19 20 DNA Artificial SequenceSynthetic DNA 19 gcaagaagtc tacaagcgcc 20 20 16 DNA Artificial SequenceSynthetic DNA 20 gccaacgtat tcacgg 16 21 20 DNA Artificial SequenceSynthetic DNA 21 tgatgaacca ctcgatcccc 20 22 20 DNA Artificial SequenceSynthetic DNA 22 aagacaccac cttctaccgc 20 23 20 DNA Artificial SequenceSynthetic DNA 23 caagtggaat tctgcagcgg 20 24 21 DNA Artificial SequenceSynthetic DNA 24 cctcctacaa aacctgctgg g 21 25 20 DNA ArtificialSequence Synthetic DNA 25 tcgcggatag cttttagggc 20 26 20 DNA ArtificialSequence Synthetic DNA 26 tgagttttta gaagactccc 20 27 20 DNA ArtificialSequence Synthetic DNA 27 cgcttcagtg gtgttgtccc 20 28 24 DNA ArtificialSequence Synthetic DNA 28 cgtaccactc cacggaaatt cccg 24 29 2369 DNABrevibacterium lactofermentum CDS (484)..(1938) 29 acagaatcag cgccggcagagaaacgtcca aagactaatc agagattcgg tataaaggta 60 aaaatcaacc tgcttaggcgtctttcgctt aaatagcgta gaatatcggg tcgatcgctt 120 ttaaacactc aggaggatccttgccggcca aaatcacgga cactcgtccc accccagaat 180 cccttcacgc tgttgaagaggaaaccgcag ccggtgcccg caggattgtt gccacctatt 240 ctaaggactt cttcgacggcgtcactttga tgtgcatgct cggcgttgaa cctcagggcc 300 tgcgttacac caaggtcgcttctgaacacg aggaagctca gccaaagaag gctacaaagc 360 ggactcgtaa ggctaccagctaagaaggct gctgctaaga aaacgaccaa gaagaccact 420 aagaaaacta ctaaaaagaccaccgcaaag aagaccacaa agaagtctta agccggatct 480 tat atg gat gat tcc aatagc ttt gta gtt gtt gct aac cgt ctg cca 528 Met Asp Asp Ser Asn Ser PheVal Val Val Ala Asn Arg Leu Pro 1 5 10 15 gtg gat atg act gtc cac ccagat ggt agc tat agc atc tcc ccc agc 576 Val Asp Met Thr Val His Pro AspGly Ser Tyr Ser Ile Ser Pro Ser 20 25 30 ccc ggt ggc ctt gtc acg ggg ctttcc ccc gtt ctg gaa caa cat cgt 624 Pro Gly Gly Leu Val Thr Gly Leu SerPro Val Leu Glu Gln His Arg 35 40 45 gga tgt tgg gtc gga tgg cct gga actgta gat gtt gca ccc gaa cca 672 Gly Cys Trp Val Gly Trp Pro Gly Thr ValAsp Val Ala Pro Glu Pro 50 55 60 ttt cga aca gat acg ggt gtt ttg ctg caccct gtt gtc ctc act gca 720 Phe Arg Thr Asp Thr Gly Val Leu Leu His ProVal Val Leu Thr Ala 65 70 75 agt gac tat gaa ggc ttc tac gag ggc ttt tcaaac gca acg ctg tgg 768 Ser Asp Tyr Glu Gly Phe Tyr Glu Gly Phe Ser AsnAla Thr Leu Trp 80 85 90 95 cct ctt ttc cac gat ctg att gtt act ccg gtgtac aac acc gat tgg 816 Pro Leu Phe His Asp Leu Ile Val Thr Pro Val TyrAsn Thr Asp Trp 100 105 110 tgg cat gcg ttt cgg gaa gta aac ctc aag ttcgct gaa gcc gtg agc 864 Trp His Ala Phe Arg Glu Val Asn Leu Lys Phe AlaGlu Ala Val Ser 115 120 125 caa gtg gcg gca cac ggt gcc act gtg tgg gtgcag gac tat cag ctg 912 Gln Val Ala Ala His Gly Ala Thr Val Trp Val GlnAsp Tyr Gln Leu 130 135 140 ttg ctg gtt cct ggc att ttg cgc cag atg cgcctt gat ttg aag atc 960 Leu Leu Val Pro Gly Ile Leu Arg Gln Met Arg LeuAsp Leu Lys Ile 145 150 155 ggt ttc ttc ctc cac att ccc ttc cct tcc cctgat ctg ttc cgt cag 1008 Gly Phe Phe Leu His Ile Pro Phe Pro Ser Pro AspLeu Phe Arg Gln 160 165 170 175 ctg ccg tgg cgt gaa gag att gtt cga ggcatg ctg ggc gca gat ttg 1056 Leu Pro Trp Arg Glu Glu Ile Val Arg Gly MetLeu Gly Ala Asp Leu 180 185 190 gtg gga ttc cat ttg gtt caa aac gca gaaaac ttc ctt gcg tta acc 1104 Val Gly Phe His Leu Val Gln Asn Ala Glu AsnPhe Leu Ala Leu Thr 195 200 205 cag cag gtt gcc ggc act gcc ggg tct catgtg ggt cag ccg gac acc 1152 Gln Gln Val Ala Gly Thr Ala Gly Ser His ValGly Gln Pro Asp Thr 210 215 220 ttg cag gtc agt ggt gaa gca ttg gtg cgtgag att ggc gct cat gtt 1200 Leu Gln Val Ser Gly Glu Ala Leu Val Arg GluIle Gly Ala His Val 225 230 235 gaa acc gct gac gga agg cga gtt agc gtcggg gcg ttc ccg atc tcg 1248 Glu Thr Ala Asp Gly Arg Arg Val Ser Val GlyAla Phe Pro Ile Ser 240 245 250 255 att gat gtt gaa atg ttt ggg gag gcgtcg aaa agc gcc gtt ctt gat 1296 Ile Asp Val Glu Met Phe Gly Glu Ala SerLys Ser Ala Val Leu Asp 260 265 270 ctt tta aaa acg ctc gac gag ccg gaaacc gta ttc ctg ggc gtt gac 1344 Leu Leu Lys Thr Leu Asp Glu Pro Glu ThrVal Phe Leu Gly Val Asp 275 280 285 cga ctg gac tac acc aag ggc att ttgcag cgc ctg ctt gcg ttt gag 1392 Arg Leu Asp Tyr Thr Lys Gly Ile Leu GlnArg Leu Leu Ala Phe Glu 290 295 300 gaa ctg ctg gaa tcc ggc gcg ttg gaggcc gac aaa gct gtg ttg ctg 1440 Glu Leu Leu Glu Ser Gly Ala Leu Glu AlaAsp Lys Ala Val Leu Leu 305 310 315 cag gtc gcg acg cct tcg cgt gag cgcatt gat cac tat cgt gtg tcg 1488 Gln Val Ala Thr Pro Ser Arg Glu Arg IleAsp His Tyr Arg Val Ser 320 325 330 335 cgt tcg cag gtc gag gaa gcc gtcggc cgt atc aat ggt cgt ttc ggt 1536 Arg Ser Gln Val Glu Glu Ala Val GlyArg Ile Asn Gly Arg Phe Gly 340 345 350 cgc atg ggg cgt ccc gtg gtg cattat cta cac agg tca ttg agc aaa 1584 Arg Met Gly Arg Pro Val Val His TyrLeu His Arg Ser Leu Ser Lys 355 360 365 aat gat ctc cag gtg ctg tat accgca gcc gat gtc atg ctg gtt acg 1632 Asn Asp Leu Gln Val Leu Tyr Thr AlaAla Asp Val Met Leu Val Thr 370 375 380 cct ttt aaa gac ggt atg aac ttggtg gct aaa gaa ttc gtg gcc aac 1680 Pro Phe Lys Asp Gly Met Asn Leu ValAla Lys Glu Phe Val Ala Asn 385 390 395 cac cgc gac ggc act ggt gct ttggtg ctg tcc gaa ttt gcc ggc gcg 1728 His Arg Asp Gly Thr Gly Ala Leu ValLeu Ser Glu Phe Ala Gly Ala 400 405 410 415 gcc act gag ctg acc ggt gcgtat tta tgc aac cca ttt gat gtg gaa 1776 Ala Thr Glu Leu Thr Gly Ala TyrLeu Cys Asn Pro Phe Asp Val Glu 420 425 430 tcc atc aaa cgg caa atg gtggca gct gtc cat gat ttg aag cac aat 1824 Ser Ile Lys Arg Gln Met Val AlaAla Val His Asp Leu Lys His Asn 435 440 445 ccg gaa tct gcg gca acg cgaatg aaa acg aac agc gag cag gtc tat 1872 Pro Glu Ser Ala Ala Thr Arg MetLys Thr Asn Ser Glu Gln Val Tyr 450 455 460 acc cac gac gtc aac gtg tgggct aat agt ttc ctg gat tgt ttg gcg 1920 Thr His Asp Val Asn Val Trp AlaAsn Ser Phe Leu Asp Cys Leu Ala 465 470 475 cag tcg gga gaa aac tcatgaaccgcgc acgaatcgcg accataggcg 1968 Gln Ser Gly Glu Asn Ser 480 485ttcttccgct tgctttactg ctggcgtcct gtggttcaga caccgtggaa atgacagatt 2028ccacctggtt ggtgaccaat atttacaccg atccagatga gtcgaattcg atcagtaatc 2088ttgtcatttc ccagcccagc ttagattttg gcaattcttc cctgtctggt ttcactggct 2148gtgtgccttt tacggggcgt gcggaattct tccaaaatgg tgagcaaagc tctgttctgg 2208atgccgatta tgtgaccttg tcttccctgg atttcgataa acttcccgat gattgccaag 2268gacaagaact caaagttcat aacgagctgg ttgatcttct gcctggttct tttgaaatct 2328ccaggacttc tggttcagaa atcttgctga ctagcgatgt c 2369 30 485 PRTBrevibacterium lactofermentum 30 Met Asp Asp Ser Asn Ser Phe Val Val ValAla Asn Arg Leu Pro Val 1 5 10 15 Asp Met Thr Val His Pro Asp Gly SerTyr Ser Ile Ser Pro Ser Pro 20 25 30 Gly Gly Leu Val Thr Gly Leu Ser ProVal Leu Glu Gln His Arg Gly 35 40 45 Cys Trp Val Gly Trp Pro Gly Thr ValAsp Val Ala Pro Glu Pro Phe 50 55 60 Arg Thr Asp Thr Gly Val Leu Leu HisPro Val Val Leu Thr Ala Ser 65 70 75 80 Asp Tyr Glu Gly Phe Tyr Glu GlyPhe Ser Asn Ala Thr Leu Trp Pro 85 90 95 Leu Phe His Asp Leu Ile Val ThrPro Val Tyr Asn Thr Asp Trp Trp 100 105 110 His Ala Phe Arg Glu Val AsnLeu Lys Phe Ala Glu Ala Val Ser Gln 115 120 125 Val Ala Ala His Gly AlaThr Val Trp Val Gln Asp Tyr Gln Leu Leu 130 135 140 Leu Val Pro Gly IleLeu Arg Gln Met Arg Leu Asp Leu Lys Ile Gly 145 150 155 160 Phe Phe LeuHis Ile Pro Phe Pro Ser Pro Asp Leu Phe Arg Gln Leu 165 170 175 Pro TrpArg Glu Glu Ile Val Arg Gly Met Leu Gly Ala Asp Leu Val 180 185 190 GlyPhe His Leu Val Gln Asn Ala Glu Asn Phe Leu Ala Leu Thr Gln 195 200 205Gln Val Ala Gly Thr Ala Gly Ser His Val Gly Gln Pro Asp Thr Leu 210 215220 Gln Val Ser Gly Glu Ala Leu Val Arg Glu Ile Gly Ala His Val Glu 225230 235 240 Thr Ala Asp Gly Arg Arg Val Ser Val Gly Ala Phe Pro Ile SerIle 245 250 255 Asp Val Glu Met Phe Gly Glu Ala Ser Lys Ser Ala Val LeuAsp Leu 260 265 270 Leu Lys Thr Leu Asp Glu Pro Glu Thr Val Phe Leu GlyVal Asp Arg 275 280 285 Leu Asp Tyr Thr Lys Gly Ile Leu Gln Arg Leu LeuAla Phe Glu Glu 290 295 300 Leu Leu Glu Ser Gly Ala Leu Glu Ala Asp LysAla Val Leu Leu Gln 305 310 315 320 Val Ala Thr Pro Ser Arg Glu Arg IleAsp His Tyr Arg Val Ser Arg 325 330 335 Ser Gln Val Glu Glu Ala Val GlyArg Ile Asn Gly Arg Phe Gly Arg 340 345 350 Met Gly Arg Pro Val Val HisTyr Leu His Arg Ser Leu Ser Lys Asn 355 360 365 Asp Leu Gln Val Leu TyrThr Ala Ala Asp Val Met Leu Val Thr Pro 370 375 380 Phe Lys Asp Gly MetAsn Leu Val Ala Lys Glu Phe Val Ala Asn His 385 390 395 400 Arg Asp GlyThr Gly Ala Leu Val Leu Ser Glu Phe Ala Gly Ala Ala 405 410 415 Thr GluLeu Thr Gly Ala Tyr Leu Cys Asn Pro Phe Asp Val Glu Ser 420 425 430 IleLys Arg Gln Met Val Ala Ala Val His Asp Leu Lys His Asn Pro 435 440 445Glu Ser Ala Ala Thr Arg Met Lys Thr Asn Ser Glu Gln Val Tyr Thr 450 455460 His Asp Val Asn Val Trp Ala Asn Ser Phe Leu Asp Cys Leu Ala Gln 465470 475 480 Ser Gly Glu Asn Ser 485 31 2956 DNA Brevibacteriumlactofermentum CDS (82)..(2514) 31 ttttcccacg cagggaaggc gtgaacactaagatcgagga cgtaccgcac gattttgcct 60 aacttttaag ggtgtttcat c atg gca cgtcca att tcc gca acg tac agg 111 Met Ala Arg Pro Ile Ser Ala Thr Tyr Arg1 5 10 ctt caa atg cga gga cct caa gca gat agc gcc ggg cgt ttc ttt ggt159 Leu Gln Met Arg Gly Pro Gln Ala Asp Ser Ala Gly Arg Phe Phe Gly 1520 25 ttt gcg cag gcc aaa gcc cag ctt ccc tat ctg aag aag cta ggc atc207 Phe Ala Gln Ala Lys Ala Gln Leu Pro Tyr Leu Lys Lys Leu Gly Ile 3035 40 agc cac ctg tac ctc tcc cct att ttt acg gcc atg cca gat tcc aat255 Ser His Leu Tyr Leu Ser Pro Ile Phe Thr Ala Met Pro Asp Ser Asn 4550 55 cat ggc tac gat gtc att gat ccc acc gcc atc aat gaa gag ctc ggt303 His Gly Tyr Asp Val Ile Asp Pro Thr Ala Ile Asn Glu Glu Leu Gly 6065 70 ggc atg gag ggt ctt cga gat ctt gct gca gct aca cac gag ttg ggc351 Gly Met Glu Gly Leu Arg Asp Leu Ala Ala Ala Thr His Glu Leu Gly 7580 85 90 atg ggc atc atc att gat att gtt ccc aac cat tta ggt gtt gcc gtt399 Met Gly Ile Ile Ile Asp Ile Val Pro Asn His Leu Gly Val Ala Val 95100 105 cca cat ttg aat cct tgg tgg tgg gat gtt cta aaa aac ggc aaa gat447 Pro His Leu Asn Pro Trp Trp Trp Asp Val Leu Lys Asn Gly Lys Asp 110115 120 tcc gct ttt gag ttc tat ttc gat att gac tgg cac gaa gac aac ggt495 Ser Ala Phe Glu Phe Tyr Phe Asp Ile Asp Trp His Glu Asp Asn Gly 125130 135 tct ggt ggc aag ctg ggc atg ccg att ctg ggt gct gaa ggc gat gaa543 Ser Gly Gly Lys Leu Gly Met Pro Ile Leu Gly Ala Glu Gly Asp Glu 140145 150 gac aag ctg gaa ttc gcg gag ctt gat gga gag aaa gtg ctc aaa tat591 Asp Lys Leu Glu Phe Ala Glu Leu Asp Gly Glu Lys Val Leu Lys Tyr 155160 165 170 ttt gac cac ctc ttc cca atc gcg cct ggt acc gaa gaa ggg acaccg 639 Phe Asp His Leu Phe Pro Ile Ala Pro Gly Thr Glu Glu Gly Thr Pro175 180 185 caa gaa gtc tac aag cgc cag cat tac cgc ctg cag ttc tgg cgcgac 687 Gln Glu Val Tyr Lys Arg Gln His Tyr Arg Leu Gln Phe Trp Arg Asp190 195 200 ggc gtg atc aac ttc cgt cgc ttc ttt tcc gtg aat acg ttg gctggc 735 Gly Val Ile Asn Phe Arg Arg Phe Phe Ser Val Asn Thr Leu Ala Gly205 210 215 atc agg caa gaa gat ccc ttg gtg ttt gaa cat act cat cgt ctgctg 783 Ile Arg Gln Glu Asp Pro Leu Val Phe Glu His Thr His Arg Leu Leu220 225 230 cgc gaa ttg gtg gcg gaa gac ctc att gac ggc gtg cgc gtc gatcac 831 Arg Glu Leu Val Ala Glu Asp Leu Ile Asp Gly Val Arg Val Asp His235 240 245 250 ccc gac ggg ctt tcc gat cct ttt gga tat ctg cac aga ctccgc gac 879 Pro Asp Gly Leu Ser Asp Pro Phe Gly Tyr Leu His Arg Leu ArgAsp 255 260 265 ctc att gga cct gac cgc tgg ctg atc atc gaa aag atc ttgagc gtt 927 Leu Ile Gly Pro Asp Arg Trp Leu Ile Ile Glu Lys Ile Leu SerVal 270 275 280 gat gaa cca ctc gat ccc cgc ctg gcc gtt gat ggc acc actggc tac 975 Asp Glu Pro Leu Asp Pro Arg Leu Ala Val Asp Gly Thr Thr GlyTyr 285 290 295 gac ccc ctc cgt gaa ctc gac ggc gtg ttt atc tcc cga gaatct gag 1023 Asp Pro Leu Arg Glu Leu Asp Gly Val Phe Ile Ser Arg Glu SerGlu 300 305 310 gac aaa ttc tcc atg ttg gcg ctg acc cac agt gga tcc acctgg gat 1071 Asp Lys Phe Ser Met Leu Ala Leu Thr His Ser Gly Ser Thr TrpAsp 315 320 325 330 gaa cgc gcc cta aaa tcc acg gag gaa agc ctc aaa cgagtc gtc gcg 1119 Glu Arg Ala Leu Lys Ser Thr Glu Glu Ser Leu Lys Arg ValVal Ala 335 340 345 caa caa gaa ctc gca gcc gaa atc tta agg ctc gcc cgcgcc atg cgc 1167 Gln Gln Glu Leu Ala Ala Glu Ile Leu Arg Leu Ala Arg AlaMet Arg 350 355 360 cgc gat aac ttc tcc acc gca ggc acc aac gtc acc gaagac aaa ctt 1215 Arg Asp Asn Phe Ser Thr Ala Gly Thr Asn Val Thr Glu AspLys Leu 365 370 375 agc gaa acc atc atc gaa tta gtc gcc gcc atg ccc gtctac cgc gcc 1263 Ser Glu Thr Ile Ile Glu Leu Val Ala Ala Met Pro Val TyrArg Ala 380 385 390 gac tac atc tcc ctc tca cgc acc acc gcc acc gtc atcgcg gag atg 1311 Asp Tyr Ile Ser Leu Ser Arg Thr Thr Ala Thr Val Ile AlaGlu Met 395 400 405 410 tcc aaa cgc ttc ccc tcc cgg cgc gac gca ctc gacctc atc tcg gcc 1359 Ser Lys Arg Phe Pro Ser Arg Arg Asp Ala Leu Asp LeuIle Ser Ala 415 420 425 gcc cta ctt ggc aat ggc gag gcc aaa atc cgc ttcgcc caa gtc tgc 1407 Ala Leu Leu Gly Asn Gly Glu Ala Lys Ile Arg Phe AlaGln Val Cys 430 435 440 ggc gcc gtc atg gcc aaa ggt gtg gaa gac acc accttc tac cgc gca 1455 Gly Ala Val Met Ala Lys Gly Val Glu Asp Thr Thr PheTyr Arg Ala 445 450 455 tct agg ctc gtt gca ctg caa gaa gtc ggt ggc gcgccg ggc agg ttc 1503 Ser Arg Leu Val Ala Leu Gln Glu Val Gly Gly Ala ProGly Arg Phe 460 465 470 ggc gtc tcc gct gca gaa ttc cac ttg ctg cag gaagaa cgc agc ctg 1551 Gly Val Ser Ala Ala Glu Phe His Leu Leu Gln Glu GluArg Ser Leu 475 480 485 490 ctg tgg cca cgc acc atg acc acc ttg tcc acgcac gac acc aaa cgc 1599 Leu Trp Pro Arg Thr Met Thr Thr Leu Ser Thr HisAsp Thr Lys Arg 495 500 505 ggc gaa gat acc cgc gcc cgc atc atc tcc ctgtcc gaa gtc ccc gat 1647 Gly Glu Asp Thr Arg Ala Arg Ile Ile Ser Leu SerGlu Val Pro Asp 510 515 520 atg tac tcc gag ctg gtc aat cgt gtt ttc gcagtg ctc ccc gcg cca 1695 Met Tyr Ser Glu Leu Val Asn Arg Val Phe Ala ValLeu Pro Ala Pro 525 530 535 gac ggc gca acg ggc agt ttc ctc cta caa aacctg ctg ggc gta tgg 1743 Asp Gly Ala Thr Gly Ser Phe Leu Leu Gln Asn LeuLeu Gly Val Trp 540 545 550 ccc gcc gac ggc gtg atc acc gat gcg ctg cgcgat cga ttc agg gaa 1791 Pro Ala Asp Gly Val Ile Thr Asp Ala Leu Arg AspArg Phe Arg Glu 555 560 565 570 tac gcc cta aaa gct atc cgc gaa gca tccaca aaa acc acg tgg gtg 1839 Tyr Ala Leu Lys Ala Ile Arg Glu Ala Ser ThrLys Thr Thr Trp Val 575 580 585 gac ccc aac gag tcc ttc gag gct gcg gtctgc gat tgg gtg gaa gcg 1887 Asp Pro Asn Glu Ser Phe Glu Ala Ala Val CysAsp Trp Val Glu Ala 590 595 600 ctt ttc gac gga ccc tcc acc tca tta atcacc gaa ttt gtc tcc cac 1935 Leu Phe Asp Gly Pro Ser Thr Ser Leu Ile ThrGlu Phe Val Ser His 605 610 615 atc aac cgt ggc tct gtg aat atc tcc ttaggt agg aaa ctg ctg caa 1983 Ile Asn Arg Gly Ser Val Asn Ile Ser Leu GlyArg Lys Leu Leu Gln 620 625 630 atg gtg ggc gct gga atc ccc gac act taccaa gga act gag ttt tta 2031 Met Val Gly Ala Gly Ile Pro Asp Thr Tyr GlnGly Thr Glu Phe Leu 635 640 645 650 gaa gac tcc ctg gta gat ccc gat aaccga cgc ttt gtt gat tac acc 2079 Glu Asp Ser Leu Val Asp Pro Asp Asn ArgArg Phe Val Asp Tyr Thr 655 660 665 gcc aga gaa caa gtc ctg gag cgc ctgcaa acc tgg gat tgg acg cag 2127 Ala Arg Glu Gln Val Leu Glu Arg Leu GlnThr Trp Asp Trp Thr Gln 670 675 680 gtt aat tcg gta gaa gac ttg gtg gataac gcc gac atc gcc aaa atg 2175 Val Asn Ser Val Glu Asp Leu Val Asp AsnAla Asp Ile Ala Lys Met 685 690 695 gcc gtg gtc cat aaa tcc ctc gag ttgcgt gct gaa ttt cgt gca agc 2223 Ala Val Val His Lys Ser Leu Glu Leu ArgAla Glu Phe Arg Ala Ser 700 705 710 ttt gtt ggt gga gat cat cag gca gtattt ggc gaa ggt cgc gca gaa 2271 Phe Val Gly Gly Asp His Gln Ala Val PheGly Glu Gly Arg Ala Glu 715 720 725 730 tcc cac atc atg ggc atc gcc cgcggt aca gac cga aac cac ctc aac 2319 Ser His Ile Met Gly Ile Ala Arg GlyThr Asp Arg Asn His Leu Asn 735 740 745 atc att gct ctt gct acc cgt cgacca ctg atc ttg gaa gac cgt ggc 2367 Ile Ile Ala Leu Ala Thr Arg Arg ProLeu Ile Leu Glu Asp Arg Gly 750 755 760 gga tgg tat gac acc acc gtc acgctt cct ggt gga caa tgg gaa gac 2415 Gly Trp Tyr Asp Thr Thr Val Thr LeuPro Gly Gly Gln Trp Glu Asp 765 770 775 agg ctc acc ggg caa cgc ttc agtggt gtt gtc cca gcc acc gat ttg 2463 Arg Leu Thr Gly Gln Arg Phe Ser GlyVal Val Pro Ala Thr Asp Leu 780 785 790 ttc tca cat tta ccc gta tct ttgttg gtt tta gta ccc gat agt gag 2511 Phe Ser His Leu Pro Val Ser Leu LeuVal Leu Val Pro Asp Ser Glu 795 800 805 810 ttt tgatccctgc acaggaaagttagcggcgct actatgaacg atcgatatgt 2564 Phe ctgacaacac tctctcccaatttggcagtt actaccacga attccgacgt gcccatccca 2624 tggccgacgt cgaattcctcctagcaattg aagaattact cacagacggt ggtgtcacct 2684 tcgatcgcgt caccacacgcatcaaagaat ggtcaagcct gaaagccaag gctcgcaagc 2744 gtcgcgacga tggctcgttgatctaccctg atccgcgcaa agacatccac gacatgatcg 2804 gtgttcggat caccacgtaccactccacgg aaattcccgt ggccttaaaa gtgctccaag 2864 actccttcat cgtccacaaatccgtagaca aagccgctga aactcgcatc tcaggcggct 2924 ttggttacgg ctcccaccaccaaggattnt ag 2956 32 811 PRT Brevibacterium lactofermentum 32 Met AlaArg Pro Ile Ser Ala Thr Tyr Arg Leu Gln Met Arg Gly Pro 1 5 10 15 GlnAla Asp Ser Ala Gly Arg Phe Phe Gly Phe Ala Gln Ala Lys Ala 20 25 30 GlnLeu Pro Tyr Leu Lys Lys Leu Gly Ile Ser His Leu Tyr Leu Ser 35 40 45 ProIle Phe Thr Ala Met Pro Asp Ser Asn His Gly Tyr Asp Val Ile 50 55 60 AspPro Thr Ala Ile Asn Glu Glu Leu Gly Gly Met Glu Gly Leu Arg 65 70 75 80Asp Leu Ala Ala Ala Thr His Glu Leu Gly Met Gly Ile Ile Ile Asp 85 90 95Ile Val Pro Asn His Leu Gly Val Ala Val Pro His Leu Asn Pro Trp 100 105110 Trp Trp Asp Val Leu Lys Asn Gly Lys Asp Ser Ala Phe Glu Phe Tyr 115120 125 Phe Asp Ile Asp Trp His Glu Asp Asn Gly Ser Gly Gly Lys Leu Gly130 135 140 Met Pro Ile Leu Gly Ala Glu Gly Asp Glu Asp Lys Leu Glu PheAla 145 150 155 160 Glu Leu Asp Gly Glu Lys Val Leu Lys Tyr Phe Asp HisLeu Phe Pro 165 170 175 Ile Ala Pro Gly Thr Glu Glu Gly Thr Pro Gln GluVal Tyr Lys Arg 180 185 190 Gln His Tyr Arg Leu Gln Phe Trp Arg Asp GlyVal Ile Asn Phe Arg 195 200 205 Arg Phe Phe Ser Val Asn Thr Leu Ala GlyIle Arg Gln Glu Asp Pro 210 215 220 Leu Val Phe Glu His Thr His Arg LeuLeu Arg Glu Leu Val Ala Glu 225 230 235 240 Asp Leu Ile Asp Gly Val ArgVal Asp His Pro Asp Gly Leu Ser Asp 245 250 255 Pro Phe Gly Tyr Leu HisArg Leu Arg Asp Leu Ile Gly Pro Asp Arg 260 265 270 Trp Leu Ile Ile GluLys Ile Leu Ser Val Asp Glu Pro Leu Asp Pro 275 280 285 Arg Leu Ala ValAsp Gly Thr Thr Gly Tyr Asp Pro Leu Arg Glu Leu 290 295 300 Asp Gly ValPhe Ile Ser Arg Glu Ser Glu Asp Lys Phe Ser Met Leu 305 310 315 320 AlaLeu Thr His Ser Gly Ser Thr Trp Asp Glu Arg Ala Leu Lys Ser 325 330 335Thr Glu Glu Ser Leu Lys Arg Val Val Ala Gln Gln Glu Leu Ala Ala 340 345350 Glu Ile Leu Arg Leu Ala Arg Ala Met Arg Arg Asp Asn Phe Ser Thr 355360 365 Ala Gly Thr Asn Val Thr Glu Asp Lys Leu Ser Glu Thr Ile Ile Glu370 375 380 Leu Val Ala Ala Met Pro Val Tyr Arg Ala Asp Tyr Ile Ser LeuSer 385 390 395 400 Arg Thr Thr Ala Thr Val Ile Ala Glu Met Ser Lys ArgPhe Pro Ser 405 410 415 Arg Arg Asp Ala Leu Asp Leu Ile Ser Ala Ala LeuLeu Gly Asn Gly 420 425 430 Glu Ala Lys Ile Arg Phe Ala Gln Val Cys GlyAla Val Met Ala Lys 435 440 445 Gly Val Glu Asp Thr Thr Phe Tyr Arg AlaSer Arg Leu Val Ala Leu 450 455 460 Gln Glu Val Gly Gly Ala Pro Gly ArgPhe Gly Val Ser Ala Ala Glu 465 470 475 480 Phe His Leu Leu Gln Glu GluArg Ser Leu Leu Trp Pro Arg Thr Met 485 490 495 Thr Thr Leu Ser Thr HisAsp Thr Lys Arg Gly Glu Asp Thr Arg Ala 500 505 510 Arg Ile Ile Ser LeuSer Glu Val Pro Asp Met Tyr Ser Glu Leu Val 515 520 525 Asn Arg Val PheAla Val Leu Pro Ala Pro Asp Gly Ala Thr Gly Ser 530 535 540 Phe Leu LeuGln Asn Leu Leu Gly Val Trp Pro Ala Asp Gly Val Ile 545 550 555 560 ThrAsp Ala Leu Arg Asp Arg Phe Arg Glu Tyr Ala Leu Lys Ala Ile 565 570 575Arg Glu Ala Ser Thr Lys Thr Thr Trp Val Asp Pro Asn Glu Ser Phe 580 585590 Glu Ala Ala Val Cys Asp Trp Val Glu Ala Leu Phe Asp Gly Pro Ser 595600 605 Thr Ser Leu Ile Thr Glu Phe Val Ser His Ile Asn Arg Gly Ser Val610 615 620 Asn Ile Ser Leu Gly Arg Lys Leu Leu Gln Met Val Gly Ala GlyIle 625 630 635 640 Pro Asp Thr Tyr Gln Gly Thr Glu Phe Leu Glu Asp SerLeu Val Asp 645 650 655 Pro Asp Asn Arg Arg Phe Val Asp Tyr Thr Ala ArgGlu Gln Val Leu 660 665 670 Glu Arg Leu Gln Thr Trp Asp Trp Thr Gln ValAsn Ser Val Glu Asp 675 680 685 Leu Val Asp Asn Ala Asp Ile Ala Lys MetAla Val Val His Lys Ser 690 695 700 Leu Glu Leu Arg Ala Glu Phe Arg AlaSer Phe Val Gly Gly Asp His 705 710 715 720 Gln Ala Val Phe Gly Glu GlyArg Ala Glu Ser His Ile Met Gly Ile 725 730 735 Ala Arg Gly Thr Asp ArgAsn His Leu Asn Ile Ile Ala Leu Ala Thr 740 745 750 Arg Arg Pro Leu IleLeu Glu Asp Arg Gly Gly Trp Tyr Asp Thr Thr 755 760 765 Val Thr Leu ProGly Gly Gln Trp Glu Asp Arg Leu Thr Gly Gln Arg 770 775 780 Phe Ser GlyVal Val Pro Ala Thr Asp Leu Phe Ser His Leu Pro Val 785 790 795 800 SerLeu Leu Val Leu Val Pro Asp Ser Glu Phe 805 810 33 30 DNA ArtificialSequence Synthetic DNA 33 ccaaaatcga taacatcaat cgagatcggg 30 34 30 DNAArtificial Sequence Synthetic DNA 34 cttgatcgat taaaaacgct cgacgagccg 30

What is claimed is:
 1. A coryneform bacterium having L-glutamic acidproducing ability, wherein trehalose synthesis ability is decreased ordeleted in the bacterium.
 2. The coryneform bacteria according to claim1, wherein the trehalose synthesis ability is decreased or deleted byintroducing a mutation into a chromosomal gene coding for an enzyme intrehalose systhesis pathway or disrupting the gene.
 3. The coryneformbacteria according to claim 2, wherein the gene coding for the enzyme intrehalose synthesis pathway consists of a gene coding fortrehalose-6-phosphate synthase, a gene coding for maltooligosyltrehalosesynthase, or both of these genes.
 4. The coryneform bacteria accordingto claim 3, wherein the gene coding for trehalose-6-phosphate synthasecodes for the amino acid sequence of SEQ ID NO: 30, and the gene codingfor maltooligosyltrehalose synthase codes for the amino acid sequence ofSEQ ID NO:
 32. 5. A method for producing L-glutamic acid comprising thesteps of culturing a coryneform bacterium according to any one of claims1-4 in a medium to produce and accumulate L-glutamic acid in the medium,and collecting the L-glutamic acid from the medium.
 6. A DNA coding fora protein defined in the following (A) or (B): (A) a protein having theamino acid sequence of SEQ ID NO: 30, (B) a protein having the aminoacid sequence of SEQ ID NO: 30 including substitution, deletion,insertion or addition of one or several amino acid residues and havingtrehalose-6-phosphate synthase activity.
 7. A DNA according to claim 6,which is a DNA defined in the following (a) or (b): (a) a DNA containinga nucleotide sequence comprising at least the residues of nucleotidenumbers 484-1938 in the nucleotide sequence of SEQ ID NO: 29, (b) a DNAhybridizable with a nucleotide sequence comprising at least the residuesof nucleotide numbers 484-1938 in the nucleotide sequence of SEQ ID NO:29 under a stringent condition, showing homology of 55% or more to theforegoing nucleotide sequence, and coding for a protein havingtrehalose-6-phosphate synthase activity.
 8. A DNA coding for a proteindefined in the following (A) or (B): (A) a protein having the amino acidsequence of SEQ ID NO: 32, (B) a protein having the amino acid sequenceof SEQ ID NO: 32 including substitution, deletion, insertion or additionof one or several amino acid residues and having maltooligosyltrehalosesynthase activity.
 9. A DNA according to claim 8, which is a DNA definedin the following (a) or (b): (a) a DNA containing a nucleotide sequencecomprising at least the residues of nucleotide numbers 82-2514 in thenucleotide sequence of SEQ ID NO: 31, (b) a DNA hybridizable with anucleotide sequence comprising at least the residues of nucleotidenumbers 82-2514 in the nucleotide sequence of SEQ ID NO: 31 under astringent condition, showing homology of 60% or more to the foregoingnucleotide sequence, and coding for a protein havingmaltooligosyltrehalose synthase activity.